The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.
22 Related JoVE Articles!
Simultaneous Long-term Recordings at Two Neuronal Processing Stages in Behaving Honeybees
Institutions: University of Würzburg.
In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent and divergent anatomical connections including feed forward and feedback wiring. Furthermore, information of the same origin is partially sent via parallel pathways to different and sometimes into the same brain areas. To understand the evolutionary benefits as well as the computational advantages of these wiring strategies and especially their temporal dependencies on each other, it is necessary to have simultaneous access to single neurons of different tracts or neuropiles in the same preparation at high temporal resolution. Here we concentrate on honeybees by demonstrating a unique extracellular long term access to record multi unit activity at two subsequent neuropiles1
, the antennal lobe (AL), the first olfactory processing stage and the mushroom body (MB), a higher order integration center involved in learning and memory formation, or two parallel neuronal tracts2
connecting the AL with the MB. The latter was chosen as an example and will be described in full. In the supporting video the construction and permanent insertion of flexible multi channel wire electrodes is demonstrated. Pairwise differential amplification of the micro wire electrode channels drastically reduces the noise and verifies that the source of the signal is closely related to the position of the electrode tip. The mechanical flexibility of the used wire electrodes allows stable invasive long term recordings over many hours up to days, which is a clear advantage compared to conventional extra and intracellular in vivo
Neuroscience, Issue 89, honeybee brain, olfaction, extracellular long term recordings, double recordings, differential wire electrodes, single unit, multi-unit recordings
A Proboscis Extension Response Protocol for Investigating Behavioral Plasticity in Insects: Application to Basic, Biomedical, and Agricultural Research
Institutions: Arizona State University.
Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g.
, food, mates, threats). There are several behavioral mechanisms through which an insect learns salient associations and relates them to these events. It is important to understand this behavioral plasticity for programs aimed toward assisting insects that are beneficial for agriculture. This understanding can also be used for discovering solutions to biomedical and agricultural problems created by insects that act as disease vectors and pests. The Proboscis Extension Response (PER) conditioning protocol was developed for honey bees (Apis mellifera
) over 50 years ago to study how they perceive and learn about floral odors, which signal the nectar and pollen resources a colony needs for survival. The PER procedure provides a robust and easy-to-employ framework for studying several different ecologically relevant mechanisms of behavioral plasticity. It is easily adaptable for use with several other insect species and other behavioral reflexes. These protocols can be readily employed in conjunction with various means for monitoring neural activity in the CNS via electrophysiology or bioimaging, or for manipulating targeted neuromodulatory pathways. It is a robust assay for rapidly detecting sub-lethal effects on behavior caused by environmental stressors, toxins or pesticides.
We show how the PER protocol is straightforward to implement using two procedures. One is suitable as a laboratory exercise for students or for quick assays of the effect of an experimental treatment. The other provides more thorough control of variables, which is important for studies of behavioral conditioning. We show how several measures for the behavioral response ranging from binary yes/no to more continuous variable like latency and duration of proboscis extension can be used to test hypotheses. And, we discuss some pitfalls that researchers commonly encounter when they use the procedure for the first time.
Neuroscience, Issue 91, PER, conditioning, honey bee, olfaction, olfactory processing, learning, memory, toxin assay
A Procedure to Observe Context-induced Renewal of Pavlovian-conditioned Alcohol-seeking Behavior in Rats
Institutions: Concordia University.
Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate drug-seeking behavior and prompt relapse in abstinent drug users. We have developed a procedure to study the behavioral and neural processes that mediate the impact of context on alcohol-seeking behavior in rats. Following acclimation to the taste and pharmacological effects of 15% ethanol in the home cage, male Long-Evans rats receive Pavlovian discrimination training (PDT) in conditioning chambers. In each daily (Mon-Fri) PDT session, 16 trials each of two different 10 sec auditory conditioned stimuli occur. During one stimulus, the CS+, 0.2 ml of 15% ethanol is delivered into a fluid port for oral consumption. The second stimulus, the CS-, is not paired with ethanol. Across sessions, entries into the fluid port during the CS+ increase, whereas entries during the CS- stabilize at a lower level, indicating that a predictive association between the CS+ and ethanol is acquired. During PDT each chamber is equipped with a specific configuration of visual, olfactory and tactile contextual stimuli. Following PDT, extinction training is conducted in the same chamber that is now equipped with a different configuration of contextual stimuli. The CS+ and CS- are presented as before, but ethanol is withheld, which causes a gradual decline in port entries during the CS+. At test, rats are placed back into the PDT context and presented with the CS+ and CS- as before, but without ethanol. This manipulation triggers a robust and selective increase in the number of port entries made during the alcohol predictive CS+, with no change in responding during the CS-. This effect, referred to as context-induced renewal, illustrates the powerful capacity of contexts associated with alcohol consumption to stimulate alcohol-seeking behavior in response to Pavlovian alcohol cues.
Behavior, Issue 91, Behavioral neuroscience, alcoholism, relapse, addiction, Pavlovian conditioning, ethanol, reinstatement, discrimination, conditioned approach
A Lateralized Odor Learning Model in Neonatal Rats for Dissecting Neural Circuitry Underpinning Memory Formation
Institutions: Faculty of Medicine, Memorial University, University of Victoria.
Rat pups during a critical postnatal period (≤ 10 days) readily form a preference for an odor that is associated with stimuli mimicking maternal care. Such a preference memory can last from hours, to days, even life-long, depending on training parameters. Early odor preference learning provides us with a model in which the critical changes for a natural form of learning occur in the olfactory circuitry. An additional feature that makes it a powerful tool for the analysis of memory processes is that early odor preference learning can be lateralized via
single naris occlusion within the critical period. This is due to the lack of mature anterior commissural connections of the olfactory hemispheres at this early age. This work outlines behavioral protocols for lateralized odor learning using nose plugs. Acute, reversible naris occlusion minimizes tissue and neuronal damages associated with long-term occlusion and more aggressive methods such as cauterization. The lateralized odor learning model permits within-animal comparison, therefore greatly reducing variance compared to between-animal designs. This method has been used successfully to probe the circuit changes in the olfactory system produced by training. Future directions include exploring molecular underpinnings of odor memory using this lateralized learning model; and correlating physiological change with memory strength and durations.
Neuroscience, Issue 90, lateralized odor learning, rats, memory, nose plug, olfactory bulb, piriform cortex, phosphorylated CREB
Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory Neurons
Institutions: International School for Advanced Studies, Consiglio Nazionale delle Ricerche, Italian Institute of Technology.
Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1
. Caged compounds are molecules made physiologically inactive by a chemical cage that can be broken by a flash of ultraviolet light. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged compounds for the study of olfactory transduction in dissociated mouse olfactory sensory neurons. The process of olfactory transduction (Figure 1) takes place in the cilia of olfactory sensory neurons, where odorant binding to receptors leads to the increase of cAMP that opens cyclic nucleotide-gated (CNG) channels2
. Ca entry through CNG channels activates Ca-activated Cl channels. We show how to dissociate neurons from the mouse olfactory epithelium3
and how to activate CNG channels or Ca-activated Cl channels by photolysis of caged cAMP4
or caged Ca5
. We use a flash lamp6,7
to apply ultraviolet flashes to the ciliary region to uncage cAMP or Ca while patch-clamp recordings are taken to measure the current in the whole-cell voltage-clamp configuration8-11
Neuroscience, Issue 55, caged compounds, caged cAMP, caged Ca, olfactory sensory neuron, olfaction, whole-cell patch-clamp, flash photolysis, flash lampc
Isolation and Culture of Human Fungiform Taste Papillae Cells
Institutions: Monell Chemical Senses Center, New York University College of Dentistry, AFB International.
Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple stimuli and complex chemical molecules contained in foods. In human, individual fungiform papillae contain from zero to as many as 20 taste buds. There is no established protocol for culturing human taste cells, although the ability to maintain taste papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Earlier studies of taste cells have been done using freshly isolated cells in primary culture, explant cultures from rodents, or semi-intact taste buds in tissue slices1,2,3,4
. Although each of these preparations has advantages, the development of long-term cultures would have provided significant benefits, particularly for studies of taste cell proliferation and differentiation. Several groups, including ours, have been interested in the development and establishment of taste cell culture models. Most attempts to culture taste cells have reported limited viability, with cells typically not lasting beyond 3-5 d5,6,7,8
. We recently reported on a successful method for the extended culture of rodent taste cells9
. We here report for the first time the establishment of an in vitro
culture system for isolated human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability. Cells displayed many molecular and physiological features characteristic of mature taste cells. Gustducin and phospholipase C β2, (PLC-β2) mRNA were detected in many cells by reverse transcriptase-polymerase chain reaction and confirmed by sequencing. Immunocytochemistry analysis demonstrated the presence of gustducin and PLC-β2 expression in cultured taste cells. Cultured human fungiform cells also exhibited increases in intracellular calcium in response to appropriate concentrations of several taste stimuli indicating that taste receptors and at least some of the signalling pathways were present. These results sufficient indicate that taste cells from adult humans can be generated and maintained for at least eight passages. Many of the cells retain physiological and biochemical characteristics of acutely isolated cells from the adult taste epithelium to support their use as a model taste system. This system will enable further studies of the processes involved in proliferation, differentiation and function of mammalian taste receptor cells in an in vitro
Human fungiform taste papillae used for establishing human fungiform cell culture were donated for research following proper informed consent under research protocols that were reviewed and approved by the IRB committee. The protocol (#0934) was approved by Schulman Associates Institutional Review Board Inc., Cincinnati, OH. Written protocol below is based on published parameters reported by Ozdener et al
Neuroscience, Issue 63, Fungiform, taste cells, cell culture, gustducin, calcium imaging, molecular biology, human fungiform papillae
Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique
Institutions: Monell Chemical Senses Center, University of Cambridge .
Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1
, Reisert & Zhao1
, originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca2+
channel (Fig. 1
). The ensuing depolarization triggers action potential generation at the cell body2-4
In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors5
and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors6
. The suction pipette technique was later adapted to also record from ORNs7,8
. Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body9
. The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.
Neuroscience, Issue 62, Olfactory receptor neurons, ORN, suction pipette technique, receptor current, action potentials, signal transduction, electrophysiology, chemoreceptors
Isolating Nasal Olfactory Stem Cells from Rodents or Humans
Institutions: Aix Marseille University, Aix Marseille University, Aix Marseille University, The Salk Institute for Biological Studies, Aix Marseille University, Aix Marseille University.
The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy.
Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell1
. Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)2-4
or neurodegenerative (e.g. Parkinson, Alzheimer) diseases4,5
. Olfactory mucosa can be used for either comparative molecular studies4,6
or in vitro
experiments on neurogenesis3,7
The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria8-10
. We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)11
Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease4
. We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma12,13
, a cochlear damage14
or in an animal models of Parkinson's disease15
In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.
Neuroscience, Issue 54, stem cell, nose, brain, neuron, cell therapy, diagnosis, sphere
Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells
Institutions: KU Leuven.
Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+
-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3
that initiate the propagation of the Ca2+
-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+
-wave propagation are provided by gap junction channels through the direct transfer of IP3
and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+
-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+
-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 μm. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+
-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.
Cellular Biology, Issue 77, Molecular Biology, Medicine, Biomedical Engineering, Biophysics, Immunology, Ophthalmology, Gap Junctions, Connexins, Connexin 43, Calcium Signaling, Ca2+, Cell Communication, Paracrine Communication, Intercellular communication, calcium wave propagation, gap junctions, hemichannels, endothelial cells, cell signaling, cell, isolation, cell culture
Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)
Institutions: University of Wuerzburg, Max Planck Institute of Neurobiology, Martinsried, Ludwig-Maximilians University of Munich.
Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca2+
indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+
indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro
. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+
indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+
indicator and a hydrophilic fluorescent dye/Ca2+
complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of ΔF/F0
Cellular Biology, Issue 75, Neurobiology, Neuroscience, Molecular Biology, Biochemistry, Biomedical Engineering, Bioengineering, Virology, Medicine, Anatomy, Physiology, Surgery, Endoplasmic Reticulum, ER, Calcium Signaling, calcium store, calcium imaging, calcium indicator, metabotropic signaling, Ca2+, neurons, cells, mouse, animal model, cell culture, targeted esterase induced dye loading, imaging
A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent
Institutions: University of Maine, Orono, University of Maine, Orono.
Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g.
by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells3-5
. Mast cell granule component and the allergic mediator β-hexosaminidase, which is released linearly in tandem with histamine from mast cells6
, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies1
. Originally published by Naal et al.1
, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here.
Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease7-11
, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function2
. In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using ε280
= 4,200 L/M/cm)12
. This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.
Immunology, Issue 81, mast cell, basophil, degranulation, RBL-2H3, triclosan, irgasan, antibacterial, β-hexosaminidase, allergy, Asthma, toxicants, ionophore, antigen, fluorescence, microplate, UV-Vis
Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb
Institutions: UT Southwestern Medical Center, Washington University in St. Louis.
The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (<1 mm3
) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.
Neuroscience, Issue 90, vomeronasal organ, accessory olfactory bulb, ex vivo, mouse, olfaction
Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates
Institutions: University of California Riverside, University of California Riverside, University of California Riverside.
Close to two decades of research has established that astrocytes in situ
and in vivo
express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+
indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+
events using confocal microscopy. In essence, a “calcium roadmap” is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.
Neuroscience, Issue 85, astrocyte, plasticity, mGluRs, neuronal Firing, electrophysiology, Gq GPCRs, Bolus-loading, calcium, microdomains, acute slices, Hippocampus, mouse
Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation
Institutions: University of Lausanne, University of Geneva.
Peter Karlson and Martin Lüscher used the term pheromone for the first time in 19591
to describe chemicals used for intra-species communication. Pheromones are volatile or non-volatile short-lived molecules2
secreted and/or contained in biological fluids3,4
, such as urine, a liquid known to be a main source of pheromones3
. Pheromonal communication is implicated in a variety of key animal modalities such as kin interactions5,6
, hierarchical organisations3
and sexual interactions7,8
and are consequently directly correlated with the survival of a given species9,10,11
. In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO)10,12
, a paired structure located at the base of the nasal cavity, and enclosed in a cartilaginous capsule. Each VNO has a tubular shape with a lumen13,14
allowing the contact with the external chemical world. The sensory neuroepithelium is principally composed of vomeronasal bipolar sensory neurons (VSNs)15
. Each VSN extends a single dendrite to the lumen ending in a large dendritic knob bearing up to 100 microvilli implicated in chemical detection16
. Numerous subpopulations of VSNs are present. They are differentiated by the chemoreceptor they express and thus possibly by the ligand(s) they recognize17,18
. Two main vomeronasal receptor families, V1Rs and V2Rs19,20,21,22
, are composed respectively by 24023
members and are expressed in separate layers of the neuroepithelium. Olfactory receptors (ORs)25
and formyl peptide receptors (FPRs)26,27
are also expressed in VSNs.
Whether or not these neuronal subpopulations use the same downstream signalling pathway for sensing pheromones is unknown. Despite a major role played by a calcium-permeable channel (TRPC2) present in the microvilli of mature neurons28
TRPC2 independent transduction channels have been suggested6,29
. Due to the high number of neuronal subpopulations and the peculiar morphology of the organ, pharmacological and physiological investigations of the signalling elements present in the VNO are complex.
Here, we present an acute tissue slice preparation of the mouse VNO for performing calcium imaging investigations. This physiological approach allows observations, in the natural environment of a living tissue, of general or individual subpopulations of VSNs previously loaded with Fura-2AM, a calcium dye. This method is also convenient for studying any GFP-tagged pheromone receptor and is adaptable for the use of other fluorescent calcium probes. As an example, we use here a VG mouse line30
, in which the translation of the pheromone V1rb2 receptor is linked to the expression of GFP by a polycistronic strategy.
Neuroscience, Issue 58, Vomeronasal organ, VNO, pheromone, calcium imaging, tissue slice preparation, floating immunohistochemistry, GFP
High-throughput Analysis of Mammalian Olfactory Receptors: Measurement of Receptor Activation via Luciferase Activity
Institutions: Monell Chemical Senses Center.
Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors to recognize thousands of odorants. Odorant ligands have been published for fewer than 6% of human receptors1-11
. This lack of data is due in part to difficulties functionally expressing these receptors in heterologous systems. Here, we describe a method for expressing the majority of the olfactory receptor family in Hana3A cells, followed by high-throughput assessment of olfactory receptor activation using a luciferase reporter assay. This assay can be used to (1) screen panels of odorants against panels of olfactory receptors; (2) confirm odorant/receptor interaction via dose response curves; and (3) compare receptor activation levels among receptor variants. In our sample data, 328 olfactory receptors were screened against 26 odorants. Odorant/receptor pairs with varying response scores were selected and tested in dose response. These data indicate that a screen is an effective method to enrich for odorant/receptor pairs that will pass a dose response experiment, i.e.
receptors that have a bona fide response to an odorant. Therefore, this high-throughput luciferase assay is an effective method to characterize olfactory receptors—an essential step toward a model of odor coding in the mammalian olfactory system.
Neuroscience, Issue 88, Firefly luciferase, Renilla Luciferase, Dual-Glo Luciferase Assay, olfaction, Olfactory receptor, Odorant, GPCR, High-throughput
Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay
Institutions: KU Leuven.
For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous Gα16
construct is co-transfected. Following ligand binding, activation of the Gα16
subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous Gα16
, which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their efficacy throughout the years in receptor deorphanization assays. However, optimization of the assay for specific receptors may remain necessary.
Cellular Biology, Issue 89, G protein-coupled receptor (GPCR), calcium mobilization assay, reverse pharmacology, deorphanization, cellular expression system, HEK293T, Fluo-4, FlexStation
Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules
Institutions: Princeton University.
The aim of de novo
protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo
protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity.
To disseminate these methods for broader use we present Protein WISDOM (http://www.proteinwisdom.org), a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.
Genetics, Issue 77, Molecular Biology, Bioengineering, Biochemistry, Biomedical Engineering, Chemical Engineering, Computational Biology, Genomics, Proteomics, Protein, Protein Binding, Computational Biology, Drug Design, optimization (mathematics), Amino Acids, Peptides, and Proteins, De novo protein and peptide design, Drug design, In silico sequence selection, Optimization, Fold specificity, Binding affinity, sequencing
An Effective Manual Deboning Method To Prepare Intact Mouse Nasal Tissue With Preserved Anatomical Organization
Institutions: University of Maryland Baltimore County.
The mammalian nose is a multi-functional organ with intricate internal structures. The nasal cavity is lined with various epithelia such as olfactory, respiratory, and squamous epithelia which differ markedly in anatomical locations, morphology, and functions. In adult mice, the nose is covered with various skull bones, limiting experimental access to internal structures, especially those in the posterior such as the main olfactory epithelium (MOE). Here we describe an effective method for obtaining almost the entire and intact nasal tissues with preserved anatomical organization. Using surgical tools under a dissecting microscope, we sequentially remove the skull bones surrounding the nasal tissue. This procedure can be performed on both paraformaldehyde-fixed and freshly dissected, skinned mouse heads. The entire deboning procedure takes about 20-30 min, which is significantly shorter than the experimental time required for conventional chemical-based decalcification. In addition, we present an easy method to remove air bubbles trapped between turbinates, which is critical for obtaining intact thin horizontal or coronal or sagittal sections from the nasal tissue preparation. Nasal tissue prepared using our method can be used for whole mount observation of the entire epithelia, as well as morphological, immunocytochemical, RNA in situ
hybridization, and physiological studies, especially in studies where region-specific examination and comparison are of interest.
Anatomy, Issue 78, Physiology, Surgery, Tissue Engineering, Nose, Olfactory Mucosa, Olfactory Receptor Neurons, Vomeronasal Organ, skull bone removal, nasal cavity, olfactory epithelium, olfactory turbinate, respiratory epithelium, vomeronasal organ, histochemistry, mouse, animal model
Analyzing Responses of Mouse Olfactory Sensory Neurons Using the Air-phase Electroolfactogram Recording
Institutions: Johns Hopkins University.
Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and other social interactions. The electroolfactogram (EOG) recording is an informative, easy to conduct, and reliable method to assay olfactory function at the level of the olfactory epithelium. Since the 1956 description of the EOG by Ottoson in frogs1
, the EOG recording has been applied in many vertebrates including salamanders, rabbits, rats, mice, and humans (reviewed by Scott and Scott-Johnson, 2002, ref. 2). The recent advances in genetic modification in mice have rekindled interest in recording the EOG for physiological characterization of olfactory function in knock-out and knock-in mice. EOG recordings have been successfully applied to demonstrate the central role of olfactory signal transduction components3-8
, and more recently to characterize the contribution of certain regulatory mechanisms to OSN responses9-12
Odorant detection occurs at the surface of the olfactory epithelium on the cilia of OSNs, where a signal transduction cascade leads to opening of ion channels, generating a current that flows into the cilia and depolarizes the membrane13
. The EOG is the negative potential recorded extracellularly at the surface of the olfactory epithelium upon odorant stimulation, resulting from a summation of the potential changes caused by individual responsive OSNs in the recording field2
. Comparison of the amplitude and kinetics of the EOG thus provide valuable information about how genetic modification and other experimental manipulations influence the molecular signaling underlying the OSN response to odor.
Here we describe an air-phase EOG recording on a preparation of mouse olfactory turbinates. Briefly, after sacrificing the mouse, the olfactory turbinates are exposed by bisecting the head along the midline and removing the septum. The turbinate preparation is then placed in the recording setup, and a recording electrode is placed at the surface of the olfactory epithelium on one of the medial turbinates. A reference electrode is electrically connected to the tissue through a buffer solution. A continuous stream of humidified air is blown over the surface of the epithelium to keep it moist. The vapor of odorant solutions is puffed into the stream of humidified air to stimulate the epithelium. Responses are recorded and digitized for further analysis.
JoVE Neuroscience, Issue 37, olfaction, electrophysiology, field potential, generator potential, EOG
Testing Drosophila Olfaction with a Y-maze Assay
Institutions: UMR-6265 CNRS, UMR-1324 INRA, Université de Bourgogne.
Detecting signals from the environment is essential for animals to ensure their survival. To this aim, they use environmental cues such as vision, mechanoreception, hearing, and chemoperception through taste, via direct contact or through olfaction, which represents the response to a volatile molecule acting at longer range. Volatile chemical molecules are very important signals for most animals in the detection of danger, a source of food, or to communicate between individuals. Drosophila melanogaster
is one of the most common biological models for scientists to explore the cellular and molecular basis of olfaction. In order to highlight olfactory abilities of this small insect, we describe a modified choice protocol based on the Y-maze test classically used with mice. Data obtained with Y-mazes give valuable information to better understand how animals deal with their perpetually changing environment. We introduce a step-by-step protocol to study the impact of odorants on fly exploratory response using this Y-maze assay.
Neuroscience, Issue 88, environmental effects (biological, animal and plant), genetics (animal and plant), life sciences, animal biology, behavioral sciences, Y-maze, olfaction, adult, choice, behavior, Drosophila melanogaster
Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor
Institutions: Stowers Institute for Medical Research, The University of Kansas School of Medicine.
The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within the same species 1,2
. These intraspecies signals, the pheromones, as well as signals from some predators 3
, activate the vomeronasal sensory neurons (VSNs) with high levels of specificity and sensitivity 4
. At least three distinct families of G-protein coupled receptors, V1R, V2R and FPR 5-14
, are expressed in VNO neurons to mediate the detection of the chemosensory cues. To understand how pheromone information is encoded by the VNO, it is critical to analyze the response profiles of individual VSNs to various stimuli and identify the specific receptors that mediate these responses.
The neuroepithelia of VNO are enclosed in a pair of vomer bones. The semi-blind tubular structure of VNO has one open end (the vomeronasal duct) connecting to the nasal cavity. VSNs extend their dendrites to the lumen part of the VNO, where the pheromone cues are in contact with the receptors expressed at the dendritic knobs. The cell bodies of the VSNs form pseudo-stratified layers with V1R and V2R expressed in the apical and basal layers respectively 6-8
. Several techniques have been utilized to monitor responses of VSNs to sensory stimuli 4,12,15-19
. Among these techniques, acute slice preparation offers several advantages. First, compared to dissociated VSNs 3,17
, slice preparations maintain the neurons in their native morphology and the dendrites of the cells stay relatively intact. Second, the cell bodies of the VSNs are easily accessible in coronal slice of the VNO to allow electrophysiology studies and imaging experiments as compared to whole epithelium and whole-mount preparations 12,20
. Third, this method can be combined with molecular cloning techniques to allow receptor identification.
Sensory stimulation elicits strong Ca2+
influx in VSNs that is indicative of receptor activation 4,21
. We thus develop transgenic mice that express G-CaMP2 in the olfactory sensory neurons, including the VSNs 15,22
. The sensitivity and the genetic nature of the probe greatly facilitate Ca2+
imaging experiments. This method has eliminated the dye loading process used in previous studies 4,21
. We also employ a ligand delivery system that enables application of various stimuli to the VNO slices. The combination of the two techniques allows us to monitor multiple neurons simultaneously in response to large numbers of stimuli. Finally, we have established a semi-automated analysis pipeline to assist image processing.
Neuroscience, Issue 58, Vomeronasal organ, VNO, pheromone, urine, slice preparation, G-CaMP2, calcium imaging
Multielectrode Array Recordings of the Vomeronasal Epithelium
Institutions: Washington University School of Medicine.
Understanding neural circuits requires methods to record from many neurons simultaneously. For in vitro
studies, one currently available technology is planar multielectrode array (MEA) recording. Here we document the use of MEAs to study the mouse vomeronasal organ (VNO), which plays an essential role in the detection of pheromones and social cues via a diverse population of sensory neurons expressing hundreds of types of receptors. Combining MEA recording with a robotic liquid handler to deliver chemical stimuli, the sensory responses of a large and diverse population of neurons can be recorded. The preparation allows us to remove the intact neuroepithelium of the VNO from the mouse and stimulate with a battery of chemicals or potential ligands while monitoring the electrical activity of the neurons for several hours. Therefore, this technique serves as a useful method for assessing ligand activity as well as exploring the properties of receptor neurons. We present the techniques needed to prepare the vomeronasal epithelium, MEA recording, and chemical stimulation.
JoVE Neuroscience, Issue 37, electrophysiology, multielectrode array, accessory olfactory system